β-glucuronidase from Ampullaria. Purification and kinetic properties

188

150

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the ␤1,3-galactosyltransferase family on the amino-terminal side and to the ␤1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[ 14 C]GlcUA but also Nacetylgalactosamine (GalNAc) from UDP-[ 3 H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both ␤1,3-GlcUA transferase and ␤1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.

Section: Fig 1 the Predicted Amino Acid Sequence Of The Putative Humentioning

confidence: 97%

mentioning

confidence: 99%

Molecular Cloning and Expression of a Human Chondroitin Synthase

Kitagawa¹,

Uyama²,

Sugahara³

2001

188

150

“…␣-TM, which was devoid of GAG and purified by anion-exchange chromatography, immunoaffinity chromatography, and gel-filtration chromatography (17), was kindly provided by the research institute, Dai-ichi (Pharmaceutical Co., Ltd., Tokyo, Japan (18) was provided by Tokyo Zouki Chemical Co. (Tokyo, Japan). Gal␤1-3Gal␤1-4Xyl was a gift from Dr. Nancy B. Schwartz (University of Chicago, Chicago, IL).…”

Section: Methodsmentioning

confidence: 99%

Demonstration of the Immature Glycosaminoglycan Tetrasaccharide Sequence GlcAβ1–3Galβ1–3Galβ1–4Xyl on Recombinant Soluble Human α-Thrombomodulin

Nadanaka

Kitagawa

Sugahara

1998

Thrombomodulin (TM), a cell surface glycoprotein, is a critical mediator of endothelial anticoagulant defenses occurring both as a chondroitin sulfate proteoglycan (␤-TM) and a protein (␣-TM) unsubstituted by chondroitin sulfate (CS), hence its description as a "part-time" proteoglycan (PG) (Fransson, L. Å. (1987)Trends Biochem. Sci. 12, 406 -411). Sugar analysis was performed on ␣-TM to investigate a possible biosynthetic mechanism for part-time PGs. Recombinant human ␣-TM, which was expressed in CHO-K1 cells, separated by anion-exchange chromatography from ␤-TM, and purified by immunoaffinity chromatography (Nawa, K., Sakano, K., Fujiwara, H., Sato, Y., Sugiyama, N., Teruuchi, T., Iwamoto, M., and Marumoto, Y. (1990) Biochem. Biophys. Res. Commun. 171, 729 -737), was used for analysis. Preliminary sugar composition analysis after acid hydrolysis showed Xyl in addition to Gal, GalNAc, GlcNAc, Man, Fuc, and Glc. O-Glycosidically-linked oligosaccharides were liberated by mild alkaline treatment and purified. The isolated oligosaccharide fraction was derivatized with a fluorophore 2-aminobenzamide (2AB), resulting in two fluorescent components, a 2AB-oligosaccharide and a putative 2AB-Glc. Based on structural analysis by a combination of sequential exoglycosidase digestion and 500-MHz 1 H NMR spectroscopy of the 2AB-oligosaccharide, the structure of the oligosaccharide was elucidated as GlcA␤1-3Gal␤1-3Gal␤1-4Xyl, which turned out to represent a glycosaminoglycan (GAG)-protein linkage region tetrasaccharide common to various PGs and was considered to be a biosynthetic intermediate of an immature GAG chain. The results may indicate that at least one class of the so-called parttime PGs bear the linkage tetrasaccharide at the GAG attachment sites and that the critical determining step or the rate-limiting step for PG biosynthesis is the transfer of the fifth sugar residue, the first hexosamine, rather than xylose. Thrombomodulin (TM)1 is an integral membrane glycoprotein expressed on endothelial cell surfaces, which binds to thrombin with high affinity, and acts as a critical mediator of endothelial anticoagulant defenses (1, 2). Studies on the anticoagulant effects of TM demonstrated that it has cofactor activity for thrombin-dependent activation of protein C, direct inhibition of fibrinogen cleavage by thrombin, and indirect enhancement of the association of antithrombin III with thrombin. Although TM is a single polypeptide, it occurs naturally in two major glycoforms, one with (␤-TM) and the other without (␣-TM) a chondroitin sulfate (CS) chain (3, 4), and hence has been termed "part-time" PG (5). Comparative studies on the anticoagulant effects of ␣-TM and ␤-TM revealed that the CS chain of TM plays an important role in regulating the anticoagulation functions. Indeed, the acceleration of the thrombin inhibition by antithrombin III is dependent on the presence of the CS moiety on TM. This moiety is not essential for protein C activation or enhanced inhibition by protein C inhibitor, but alters thrombin conformati...

“…⌬ 4,5 -Glycuronate-2-sulfatase (EC 3.1.6.-), abbreviated as ⌬hexuronate-2-sulfatase, was purified from Flavobacterium heparinum (19). ␤-Glucuronidase (EC 3.2.1.31) purified to homogeneity from Ampullaria (freshwater apple shell) hepatopancreas (20) was obtained from Tokyo Zouki Chemical Co., Tokyo. Human liver ␣-iduronidase (EC 3.2.1.76) was a gift from Dr. J. J. Hopwood, Adelaide Children's Hospital (21).…”

Section: Methodsmentioning

confidence: 99%

A Major Common Trisulfated Hexasaccharide Core Sequence, Hexuronic Acid(2-Sulfate)-Glucosamine(N-Sulfate)-Iduronic Acid-N-Acetylglucosamine-Glucuronic Acid-Glucosamine(N-Sulfate), Isolated from the Low Sulfated Irregular Region of Porcine Intestinal Heparin

Yamada

Yamane

Tsuda

et al. 1998

The major structure of the low sulfated irregular region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography. Structures of a tetrasaccharide, a pentasaccharide, and eight hexasaccharide components in this fraction, which accounted for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, were determined by chemical and enzymatic analyses as well as 1 H NMR spectroscopy. Five compounds including one penta-and four hexasaccharides had hitherto unreported structures. The structure of the pentasaccharide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chain and may represent the reducing terminus exposed by a tissue endo-␤-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin. Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA␣1-4Glc-NAc␣1-4GlcA␤1-, and its reverse sequence, -4GlcA␤1-4GlcNAc␣1-4IdceA␣1-, was not found. The latter has not been reported to date for heparin/heparan sulfate, indicating the substrate specificity of the D-glucuronyl C-5 epimerase. Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence ⌬HexA(2-sulfate) ␣1-4GlcN(N-sulfate)␣1-4IdceA␣1-4Gl-cNAc␣1-4GlcA␤1-4GlcN(N-sulfate)